The invention relates to a method for the treatment of transgenic plants, especially crop plants that are designed to express pesticidally effective proteins.
Transgenic plants have had a significant impact on commercial agriculture, with promising benefits but raising new pest management issues. Notably, agricultural crops based on plants that have been modified with pesticidally effective crystal protein genes to express pesticidally effective proteins hold the promise of selectively targeting pests by making their food source (the plant tissues) toxic thereby reducing or eliminating the amount of chemical insecticides that are used to control pest populations.
Certain members of the gram positive bacteria belonging to the genus Bacillus produce proteins that are insecticidal. The most well characterized are those of Bacillus thuringiensis (BT), where the insecticidal proteins are found as crystalline bodies with sporulating bacteria. The insecticidal crystal proteins are characterised by their potency and specificity towards specific insect pests, many of which are agronomically important, and their relative safety to non-target insect species and vertebrates, particularly humans. They have enjoyed a long history of use in horticultural industries where the mixture of crystals and spores are sprayed just like a chemical pesticide, but they have not been used with much success on broad-acre field crops.
The insecticidal crystals are composed of a large protein that is essentially inactive. When a caterpillar ingests some of the insecticidal crystals, the alkaline reducing conditions of the insects midgut cause the crystals to dissociate and release the crystal protein. At this stage the protein toxin is inactive, but specific proteases within the gastric juices of the insect chop the protein down to its protease resistant core that is now fully active. This activated insecticidal protein then binds to a specific receptor on the brush border membranes of the cells lining the midgut and inserts itself into the cells membrane. When about eight of these aggregate together, they form a pore or channel through the membrane, and allow the cell contents to leak out causing the death of the cells essential for nutrient absorption. The insects rapidly stop feeding and eventually starve to death or die from secondary bacterial infections within about 24 hours. The processes of crystal solublization, proteolytic processing to an active insecticidal protein and the binding to a specific receptor, all make the BT proteins highly specific and very desirable from an environmental perspective.
Many thousands of different isolates of B. thuringiensis have been collected and their insecticidal protein content and activity spectrums determined. A large number of BT-insecticidal protein genes have also now been cloned and sequenced. To avoid any confusion researchers have proposed a uniform naming system for the crystal protein genes (Cry genes) based on their protein sequence and the types of insects for which they were toxic.
The proteins encoded by the Cry I genes are all similar in protein sequence and are toxic only to caterpillars, ie. the larvae of moths and butterflies (Lepidoptera); the Cry II genes encode proteins toxic to Lepidoptera and/or Diptera (flies and mosquitoes) while the Cry IV proteins are only active against Diptera. Cry III genes produce proteins active against beetle (Coleoptera) larvae. Within these major groupings smaller divisions have been made by considering the similarities or differences between the different protein sequences. The Cry I group, for example was originally divided into Cry IA, IB and IC (although it is now up to Cry IG) where the different sub-groups may only be 50% identical at the protein sequence level. Finer sub-divisions have also been made and Cry IA now consists of Cry IA(a), IA(b) and IA(c).
A number of methods have been developed for introducing desired genes into crop plant cells and growing fertile plants therefrom. See, U.S. Pat. No. 6,329,574 and http://www.cotton.pi.csiro.au/publicat/pest/transgen.htm, the contents of which are herein incorporated by reference. The method of choice depends in part upon the target species, but cotton modification often rely on a natural gene transfer agent that has evolved its own method of plant genetic engineering. The disease called Crown Gall is a plant tumor disease caused by the soil-borne bacterial pathogen, Agrobacterium tumefaciens. In the early 70""s it was recognized that the bacterium caused the disease by transferring some of its own genetic material into the DNA of the plant cells that it infected. These parasitic genes subverted the normal biochemical machinery of the infected cells and caused them to make novel compounds that only the bacterium could utilize. This process of genetic colonization by the bacterium was just what genetic engineers were looking for, provided that they could stop the bacterium causing the disease symptoms. After further study scientists were able to identify which genes caused the disease and because bacteria are much simpler organisms to genetically manipulate, they were able to replace the disease-causing genes with the novel genes they were constructing from parts of potentially useful genes. The bacterium could then be used to piggy-back genes from the test-tube into plant cells. However not all plant cells exposed to the bacterium eventually receive the novel genes. Removal of unmodified plant cells from modified cells uses antibiotic purification.
Plant cells are sensitive to many of the antibiotics that are used to control bacterial infections in animals and humans. If a gene could be isolated that gave the plant cells tolerance to one of these toxic antibiotics then if physically linked to some desirable gene and inserted into the Agrobacterium, it would provide a useful selection system to kill off those cells that don""t receive the genes during the xe2x80x9cinfectionxe2x80x9d process. Genes have been known in bacteria for many years that give the bacteria resistance to antibiotics by producing enzymes that breakdown or chemically modify the antibiotic so that it is no longer toxic. Using the techniques of gene splicing described above researchers have been able to modify a bacterial gene that encodes an enzyme that detoxifies the antibiotic kanamycin and have produced a new hybrid gene that causes the production of this enzyme in plant cells and prevents their death in the presence of potentially lethal doses of kanamycin. Combining this antibiotic selection system with plant tissue culture procedures it has been possible to use Agrobacterium to deliver genes into a wide variety of plants from petunias to cottons.
Cotton is a crop of particular interest. Commercially available forms of transgenic cotton use the CryIAc (BOLLGARD(trademark) by Monsanto) or a combination of CryIAc with Cry2Ab (BOLLGARD(trademark) II by Monsanto) genes to express the endotoxin protein of B. thuringiensis. Field efficacy reports indicate a 50-70% reduction in the amount of applied pesticide needed to control the pests Helicoverpa armigera and H. punctigera. Also of interest are crop plants modofied with the B. thuringiensis crystal toxin genes designated cryET33 and cryET34 which encode the colepteran-toxic crystal proteins, CryET33 (29-kDa) crystal protein, and the cryET34 gene encodes the 14-kDa CryET34 crystal protein. The CryET33 and CryET34 crystal proteins are toxic to red flour beetle larvae and Japanese beetle larvae. (See, U.S. Pat. No. 6,399,330.)
The use of transgenic crop plants raises new issues in the ongoing struggle towards integrated pest management. Some of these issues concern a reduction in the amount of expressed endotoxin as the plants mature which leads to a loss of efficacy in the latter stages of the growing season (the last ⅓ of the cotton growing season) and the increased probability of surviving pests that can develop immunity to the endotoxin. Such drawbacks have lead to the development of pest control strategies that dictate a planting xe2x80x9cwindowxe2x80x9d relative to the development cycle of local pests and designated pest population minimum threshold values for pesticide application.
Physiological stress and physical damage to the transgenic plants can also result in a reduction of expressed endotoxin protein with a corresponding drop in pest control efficacy. Thus, an extended drought and/or high temperatures can reduce the endotoxin expression rate in the transgenic crop and provide a significant drop in pest protection that can dictate the need for pesticide spraying.
The specific reasons for the drop in endotoxin protein expression are not well understood. In BT cotton, it is theorized that expression of the CryIAc gene drops because the CMV35S promoter concentration declines, the gene is xe2x80x9csilenced, or other post-transcription events. It is also thought that the CryIAc protein is reduced due to increased turnover, sequestration within the plant, or dilution due to growth and aging. It is understood that CryIAc transription levels are unstable in both immature and mature BT cotton plants.
It would be desirable to have a system for treating transgenic plants designed to express pesticidally effective proteins that would promote the expression of these proteins despite increasing plant maturity, physiological stress, and physical damage.
It is an objective of the invention to provide a method for treating transgenic plants, preferably transgenic crops that express pesticidal proteins, and especially for transgenic crops that express insecticidal proteins.
It is another objective of the invention to provide a method for extending the period over which expressed proteins are present in sufficient quantity to control pest insect populations feeding on the treated plants.
In accordance with these and other objectives that will become apparent from the description herein, a method for treating transgenic crop plants according to the invention comprises applying to foliage of transgenic plants that are designed to express pesticidally effective proteins a protein transport enhancer that promotes the expression and/or stability of pesticidally effective proteins within the treated plants.
Although not wishing to be bound by any particular theory of operation, it is thought that the protein transport enhancer acts in one or more of several ways: (a) as a form of protective water substitute for cellular membranes during times of water deprivation stress, (b) as a protein stabilizer for the desired pesticidal protein, and/or (c) as a binder for proteins that facilitates movement via intraplant transport mechanisms. The result is that transgenic crop plants treated according to the invention express and move pesticidally effective proteins into fruit tissues despite physiological stress from water shortage and plant damage. It is thought that the treatment according to the invention will also continue to express effective levels of pesticidal protein through plant growth and maturity.